Protein Deficiency
Proteins are synthesized intracellularly according to the genomic nucleotide sequence of a particular gene through transcription, translation, and other processes. Protein deficiency can be caused by a mutation in the coding gene, which results in (i) non-synthesis of the protein; (ii) synthesis of the protein which lacks biological activity; or (iii) synthesis of the protein containing normal or partial biological activity, but which cannot be appropriately processed to reach the native compartment of the protein. Protein deficiency disorders that result from genetic mutations are also referred to as genetic disorders.
In addition to protein deficiencies resulting from genetic mutations, some protein deficiencies arise due to a disease, or as a side effect of a treatment for a disease (e.g., chemotherapy) or as a result of nutritional insufficiency.
Current Therapies.
There are numerous disorders resulting from protein deficiencies, some of which result from mutated, misfolded proteins (conformational disorders-see infra). One current therapy for treating protein deficiencies is protein replacement therapy, which typically involves intravenous, subcutaneous or intramuscular infusion of a purified form of the corresponding wild-type protein, or implantation of the protein in a bio-erodable solid form for extended-release. One of the main complications with protein replacement therapy is attainment and maintenance of therapeutically effective amounts of protein due to rapid degradation of the infused protein. The current approach to overcome this problem is to perform numerous costly high dose infusions.
Protein replacement therapy has several additional caveats, such as difficulties with large-scale generation, purification and storage of properly folded protein, obtaining glycosylated native protein, generation of an anti-protein immune response, and inability of protein to cross the blood-brain barrier in diseases having significant central nervous system involvement.
Gene therapy using recombinant vectors containing nucleic acid sequences that encode a functional protein, or genetically modified human cells that express a functional protein, is also being used to treat protein deficiencies and other disorders that benefit from protein replacement. Although promising, this approach is also limited by technical difficulties such as the inability of vectors to infect or transduce dividing cells, low expression of the target gene, and regulation of expression once the gene is delivered.
A third, relatively recent approach to treating protein deficiencies involves the use of small molecule inhibitors to reduce the natural substrate of deficient enzyme proteins, thereby ameliorating the pathology. This “substrate deprivation” approach has been specifically described for a class of about 40 related enzyme disorders called lysosomal storage disorders or glycosphingolipid storage disorders. These heritable disorders are characterized by deficiencies in lysosomal enzymes that catalyze the breakdown of glycolipids in cells, resulting in an abnormal accumulation of lipids which disrupts cellular function. The small molecule inhibitors proposed for use as therapy are specific for inhibiting the enzymes involved in synthesis of glycolipids, reducing the amount of cellular glycolipid that needs to be broken down by the deficient enzyme. This approach is also limited in that glycolipids are necessary for biological function, and excess deprivation may cause adverse effects. Specifically, glycolipids are used by the brain to send signals from the gangliosides of neurons to another. If there are too few or too many glycolipids, the ability of the neuron to send signals is impeded.
A fourth approach, discussed below as specific chaperone strategy, rescues mutant proteins from degradation in the endoplasmic reticulum.
Protein Processing in the Endoplasmic Reticulum
Proteins are synthesized in the cytoplasm, and the newly synthesized proteins are secreted into the lumen of the endoplasmic reticulum (ER) in a largely unfolded state. In general, protein folding is governed by the principle of self assembly. Newly synthesized polypeptides fold into their native conformation based on their amino acid sequences (Anfinsen et al., Adv. Protein Chem. 1975; 29:205-300). In vivo, protein folding is complicated, because the combination of ambient temperature and high protein concentration stimulates the process of aggregation, in which amino acids normally buried in the hydrophobic core interact with their neighbors non-specifically. To avoid this problem, protein folding is usually facilitated by a special group of proteins called molecular chaperones which prevent nascent polypeptide chains from aggregating, and bind to unfolded protein such that the protein refolds in the native conformation (Hartl, Nature 1996; 381:571-580).
Molecular chaperones are present in virtually all types of cells and in most cellular compartments. Some are involved in the transport of proteins and permit cells to survive under stresses such as heat shock and glucose starvation (Gething et al., Nature 1992; 355:33-45; Caplan, Trends Cell. Biol. 1999; 9:262-268; Lin et al., Mol. Biol. Cell. 1993; 4:109-1119; Bergeron et al., Trends Biochem. Sci. 1994; 19:124-128). Among the molecular chaperones, Bip (immunoglobulin heavy-chain binding protein, Grp78) is the best characterized chaperone of the ER (Haas, Curr. Top. Microbiol. Immunol. 1991; 167:71-82). Like other molecular chaperones, Bip interacts with many secretory and membrane proteins within the ER throughout their maturation, although the interaction is normally weak and short-lived when the folding proceeds smoothly. Once the native protein conformation is achieved, the molecular chaperone no longer interacts with the protein. Bip binding to a protein that fails to fold, assemble or be properly glycosylated, becomes stable, and leads to degradation of the protein through the ER-associated degradation pathway. This process serves as a “quality control” system in the ER, ensuring that only those properly folded and assembled proteins are transported out of the ER for further maturation, and improperly folded proteins are retained for subsequent degradation (Hurtley et al., Annu. Rev. Cell. Biol. 1989; 5:277-307).
Certain DNA mutations result in amino acid substitutions that further impede, and in many cases preclude, proper folding of the mutant proteins. To correct these misfoldings, investigators have attempted to use various molecules. High concentrations of glycerol, dimethylsulfoxide (DMSO), trimethylamine N-oxide (TMAO), or deuterated water have been shown to suppress the degradation pathway and increase the intracellular trafficking of mutant protein in several diseases (Brown et al., Cell Stress Chaperones 1996; 1:117-125; Burrows et al., Proc. Natl. Acad. Sci. USA. 2000; 97:1796-801). These compounds are considered non-specific chemical chaperones to improve the general protein folding, although the mechanism of the function is still unknown. The high doses of this class of compounds required for efficacy makes them difficult or inappropriate to use clinically, although they are useful for the biochemical examination of folding defect of a protein intracellularly. These compounds also lack specificity.
Specific Chaperone Strategy
Previous patents and publications described a therapeutic strategy for rescuing endogenous enzyme proteins, specifically misfolded lysosomal enzymes, from degradation by the ER quality control machinery. This strategy employs small molecule reversible competitive inhibitors specific for a defective lysosomal enzyme associated with a particular lysosomal disorder. The strategy is as follows: since the mutant enzyme protein folds improperly in the ER (Ishii et al., Biochem. Biophys. Res. Comm. 1996; 220: 812-815), the enzyme protein is retarded in the normal transport pathway (ER→Golgi apparatus→endosome→lysosome) and rapidly degraded. Therefore, a functional compound which facilitates the correct folding of a mutant protein will serve as a site-specific chaperone for the mutant protein to promote the smooth escape from the ER quality control system. Since some inhibitors of an enzyme are known to occupy the catalytic center of enzyme, resulting in stabilization of its conformation in vitro. These specific chaperones may be designated active site-specific chaperones (ASSC).
The strategy has been specifically demonstrated for enzymes involved in the lysosomal storage disorders in U.S. Pat. Nos. 6,274,597, 6,583,158, 6,589,964, and 6,599,919, to Fan et al., and in pending U.S. application Ser. No. 10/304,396 filed Nov. 26, 2002, which are hereby incorporated herein by reference in their entirety. For example, a small molecule derivative of galactose, 1-deoxygalactonojirimycin (DGJ), a potent competitive inhibitor of the mutant Fabry enzyme α-galactosidase A (α-Gal A), effectively increased in vitro stability of a mutant α-Gal A (R301Q) at neutral pH and enhanced the mutant enzyme activity in lymphoblasts established from Fabry patients with R301Q or Q279E mutations. Furthermore, oral administration of DGJ to transgenic mice overexpressing a mutant (R301Q) α-Gal A substantially elevated the enzyme activity in major organs (Fan et al., Nature Med. 1999; 5: 112-115). Successful rescue of a misfolded protein depends on achieving a concentration of the specific inhibitor in vivo that is lower than necessary to completely inhibit the enzyme, in contrast to the substrate deprivation approach in which enzyme inhibitory concentrations are required.
In addition to the lysosomal storage disorders, a large and diverse number of diseases are now recognized as conformational diseases that are caused by adoption of non-native protein conformations, which may lead to retardation of the protein in the ER and ultimate degradation of the proteins (Kuznetsov et al., N. Engl. J. Med. 1998; 339:1688-1695; Thomas et al., Trends Biochem. Sci. 1995; 20:456-459; Bychkova et al., FEBS Lett. 1995; 359:6-8; Brooks, FEBS Lett. 1997; 409:115-120). ASSC's have been shown to rescue expression of mutant proteins other than enzymes. For example, small synthetic compounds were found to stabilize the DNA binding domain of mutant forms of the tumor suppressor protein p53, thereby allowing the protein to maintain an active conformation (Foster et al., Science 1999; 286:2507-10). Synthesis of receptors has been shown to be rescued by small molecule receptor antagonists and ligands (Morello et al., J. Clin. Invest. 2000; 105: 887-95; Petaja-Repo et al., EMBO J. 2002; 21:1628-37.) Even pharmacological rescue of membrane channel proteins and other plasma membrane transporters has been demonstrated using channel-blocking drugs or substrates (Rajamani et al., Circulation 2002; 105:2830-5; Zhou et al., J. Biol. Chem. 1999; 274:31123-26; Loo et al., J. Biol. Chem 1997; 272: 709-12). All of the above references indicate that ASSC's are capable of specific rescue of mutant proteins including, but not limited to, enzymes, receptors, membrane channel proteins, and DNA transcription factors.
In addition to mutant proteins, ASSC's have also been shown to stabilize wild-type proteins, resulting in their enhanced production and stability. As one example, it has been demonstrated that a specific ASSC, DGJ, is able to increase the amount and activity of wild-type α-Gal A in COS-7 cells transfected with a vector coding the wild-type α-Gal A sequence. The ASSC rescues the overexpressed wild-type enzyme, which is otherwise retarded in the ER quality control system, because overexpression and over production of the enzyme in the COS-7 cells exceeds the capacity of the system and leads to aggregation and degradation (see U.S. application Ser. No. 10/377,179, filed Feb. 28, 2003).
In summary, there is a need in the art for methods of improving the biological and cost efficiency of protein replacement therapy, such as for the treatment of protein deficiencies or other disorders whereby replacement proteins are administered.